Help with high Ct values for Real-Time PCR - (Jan/05/2012 )

Hi,

I am relativiely new to Real-Time PCR and need some help. I am using mRNA extracted from rat liver that I have reverse transcribed to cDNA. I have used this cDNA before (SYBER GREEN system) on another gene and everything worked fine (Ct around 21). This new gene is giving Ct values around 30 with primers I designed. I would like to get those values lower. I know that I could increase the amount of cDNA but when I do, my reference gene's (B-actin) Ct gets too low (Ct around 15). I would like to avoid this if possible so that I can still use the Pfaffl method for analysis. Or can I use the Pfaffl method on different amounts of cDNA (we're talking like orders of magnitude different).

Any suggestions would be very helpful. Thanks.

-PCB2010-

To have your new gene's primers concentration at high (e. g. 900nM) while the actin primers at low (e. g. 250nM).